Calcium Binding Promotes Prion Protein Fragment 90–231 Conformational Change toward a Membrane Destabilizing and Cytotoxic Structure

Abstract

The pathological form of prion protein (PrPSc), as other amyloidogenic proteins, causes a marked increase of membrane permeability. PrPSc extracted from infected Syrian hamster brains induces a considerable change in membrane ionic conductance, although the contribution of this interaction to the molecular mechanism of neurodegeneration process is still controversial. We previously showed that the human PrP fragment 90–231 (hPrP90–231) increases ionic conductance across artificial lipid bilayer, in a calcium-dependent manner, producing an alteration similar to that observed for PrPSc. In the present study we demonstrate that hPrP90–231, pre-incubated with 10 mM Ca++ and then re-suspended in physiological external solution increases not only membrane conductance but neurotoxicity as well. Furthermore we show the existence of a direct link between these two effects as demonstrated by a highly statistically significant correlation in several experimental conditions. A similar correlation between increased membrane conductance and cell degeneration has been observed assaying hPrP90–231 bearing pathogenic mutations (D202N and E200K). We also report that Ca++ binding to hPrP90–231 induces a conformational change based on an alteration of secondary structure characterized by loss of alpha-helix content causing hydrophobic amino acid exposure and proteinase K resistance. These features, either acquired after controlled thermal denaturation or induced by D202N and E200K mutations were previously identified as responsible for hPrP90–231 cytotoxicity. Finally, by in silico structural analysis, we propose that Ca++ binding to hPrP90–231 modifies amino acid orientation, in the same way induced by E200K mutation, thus suggesting a pathway for the structural alterations responsible of PrP neurotoxicity.

Multiplexed Isobaric Tag‐Based Profiling of Seven Murine Tissues Following In Vivo Nicotine Treatment Using a Minimalistic Proteomics Strategy

“…Similarly, in K562 cells, exosome release is regulated by a calcium-dependent mechanism [43] . In addition, calcium is also related to prion protein processing, for example, calcium binding promotes prion protein fragment 90-231 conformational change [44] . Moreover, α7 nAChR-mediated calcium influx may have a role in calcium signaling induced by prion protein interaction with stress-inducible protein 1 [45] .…”

Mutant PrPCJD prevails over wild-type PrPCJD in the brain of V210I and R208H genetic Creutzfeldt–Jakob disease patients

“…The results of Western blotting and silver staining of SDS-polyacrylamide gels showed that the procedure adopted to process brain samples removes the vast majority of cellular proteins yielding a fraction highly enriched in PrP [27][28][29][30] . However, residual traces of non-PrP factors detected in these samples [17] could influence the chromatographic behaviour, the ionization efficiency of PrP peptides or both [18], causing any quantitative measurements of relevant PrP peptides poorly reliable.…”

“…A third mechanism envisages that V210I and R208H mutations facilitate the interaction between mutant PrP C and other organic [17,26] or inorganic [27,28] factors, which might be involved in the formation of the early steps of polymerization [29].…”

“…Synthetic, non-deuterated (ERVVEQMA, EHVVEQMA, and ERVIEQMA, used as calibrants) and deuterated peptides (ERV d8-VEQMA, EHV d8 VEQMA, and ERV d8 IEQMA, used as internal standards) (Inbios, Naples, Italy) were treated with CNBr to obtain 207-213 peptides with C-terminus identical to CNBr-treated PrP [27][28][29][30] . Hereby, the C-ter homoserine lactone of these peptides is indicated with a methionine residue (abbreviated with a M).…”

“…Equal amounts of CNBr-treated wildtype and 210I or 208H mutant deuterated peptides were dissolved in 50 ll of 0.056% TFA and added to the corresponding PrP [27][28][29][30] sample as internal standards to determine the retention times of the corresponding PrP 27-30 -derived peptides.…”